After treatment, cells were harvested and the ATP levels were determined using the ATPLite™ assay kit obtained from PerkinElmer (Boston, MA) following the protocol provided by the manufacturer. Cell culture media were collected for the determination of glycolysis activity with a cell-based assay kit, which was designed to detect extracellular levels of L-lactate, the end-product of cellular glycolysis (Catalog #600450, Cayman Chemical, Ann Arbor, MI).
For HPLC-based measurement, cells were harvested, washed, and re-suspended in PBS. Nucleotides (ATP and AMP) were extracted by fast lysing the cells in 0.05 M KOH solution, and then immediately neutralized to pH 6 with 0.1 M KH2PO4. After centrifuge, the supernatant was analyzed by a gradient HPLC method on a Waters e2695 HPLC with UV detection at 254 nm and 340 nm (Waters e2,489 diode array UV detector, Waters, MA). The reversed-phase chromatography was performed with an XBridge™ C18 column 3.5 μm (Waters, Milford, MA). Mobile phase (pH 6) contained acetonitrile (2% for Solvent A and 30% for Solvent B), 0.1 M KH2PO4, and 0.008 M Tetrabutylammonium hydrogen sulfate. The Empower II software (Waters, MA) was used for instrument control and data analysis. All values were normalized to the protein content of whole homogenaties using the bicinchoninic acid method (Pierce Biotechnology, IL).
For HPLC-based measurement, cells were harvested, washed, and re-suspended in PBS. Nucleotides (ATP and AMP) were extracted by fast lysing the cells in 0.05 M KOH solution, and then immediately neutralized to pH 6 with 0.1 M KH2PO4. After centrifuge, the supernatant was analyzed by a gradient HPLC method on a Waters e2695 HPLC with UV detection at 254 nm and 340 nm (Waters e2,489 diode array UV detector, Waters, MA). The reversed-phase chromatography was performed with an XBridge™ C18 column 3.5 μm (Waters, Milford, MA). Mobile phase (pH 6) contained acetonitrile (2% for Solvent A and 30% for Solvent B), 0.1 M KH2PO4, and 0.008 M Tetrabutylammonium hydrogen sulfate. The Empower II software (Waters, MA) was used for instrument control and data analysis. All values were normalized to the protein content of whole homogenaties using the bicinchoninic acid method (Pierce Biotechnology, IL).